Background454 sequencing technology is a promising approach for characterizing HIV-1 populations and for identifying low frequency mutations. The utility of 454 technology for determining allele frequencies and linkage associations in HIV infected individuals has not been extensively investigated. We evaluated the performance of 454 sequencing for characterizing HIV populations with defined allele frequencies.ResultsWe constructed two HIV-1 RT clones. Clone A was a wild type sequence. Clone B was identical to clone A except it contained 13 introduced drug resistant mutations. The clones were mixed at ratios ranging from 1% to 50% and were amplified by standard PCR conditions and by PCR conditions aimed at reducing PCR-based recombination. The products were sequenced using 454 pyrosequencing. Sequence analysis from standard PCR amplification revealed that 14% of all sequencing reads from a sample with a 50:50 mixture of wild type and mutant DNA were recombinants. The majority of the recombinants were the result of a single crossover event which can happen during PCR when the DNA polymerase terminates synthesis prematurely. The incompletely extended template then competes for primer sites in subsequent rounds of PCR. Although less often, a spectrum of other distinct crossover patterns was also detected. In addition, we observed point mutation errors ranging from 0.01% to 1.0% per base as well as indel (insertion and deletion) errors ranging from 0.02% to nearly 50%. The point errors (single nucleotide substitution errors) were mainly introduced during PCR while indels were the result of pyrosequencing. We then used new PCR conditions designed to reduce PCR-based recombination. Using these new conditions, the frequency of recombination was reduced 27-fold. The new conditions had no effect on point mutation errors. We found that 454 pyrosequencing was capable of identifying minority HIV-1 mutations at frequencies down to 0.1% at some nucleotide positions.ConclusionStandard PCR amplification results in a high frequency of PCR-introduced recombination precluding its use for linkage analysis of HIV populations using 454 pyrosequencing. We designed a new PCR protocol that resulted in a much lower recombination frequency and provided a powerful technique for linkage analysis and haplotype determination in HIV-1 populations. Our analyses of 454 sequencing results also demonstrated that at some specific HIV-1 drug resistant sites, mutations can reliably be detected at frequencies down to 0.1%.

Single-dose nevirapine (sdNVP) for prevention of mother-to-child transmission of HIV-1 can select nevirapine (NVP)-resistant variants, but the frequency, duration, and clinical significance of this resistance is not well defined. We used a sensitive allele-specific PCR assay to assess the emergence and persistence of NVPresistant variants in plasma samples from 22 women with HIV-1 subtype C infection who participated in a study of sdNVP for prevention of mother-to-child transmission of HIV-1. The women were categorized into three groups on the basis of detection of NVP resistance by standard genotype analysis. Group 1 (n ‫؍‬ 6) had NVP resistance detected at 2 and 6 mo after sdNVP, but not at 12 mo. Group 2 (n ‫؍‬ 9) had NVP resistance detected at 2 mo, but not 6 mo. Group 3 (n ‫؍‬ 7) had no NVP resistance detected at any time point. Allele-specific PCR analysis for the two most common NVP resistance mutations (K103N and Y181C) detected NVP-resistant variants in most (16 of 21) samples that were negative for NVP resistance by standard genotype, at levels ranging from 0.1% to 20% 1 yr after treatment. The frequency of NVP-resistant mutations decreased over time, but persisted above predose levels for more than 1 yr in >23% of the women. These findings highlight the urgent need for studies assessing the impact of sdNVP on the efficacy of subsequent antiretroviral therapy containing NVP or other nonnucleoside reverse transcriptase inhibitors.allele-specific PCR ͉ resistance ͉ low-frequency mutants T he treatment of pregnant women with antiretroviral therapy, in combination with zidovudine chemoprophylaxis administered to newborns, has dramatically reduced vertical transmission of HIV in developed countries (1-5). However, perinatal transmission of HIV-1 remains a major problem in developing countries because of limited access to antiretroviral therapy (6-8). In this latter setting, the simple regimen of maternal single-dose intrapartum nevirapine (NVP) and single-dose NVP (sdNVP) for the newborn reduces the risk of mother-to-child HIV-1 transmission (MTCT) by 41% through age 18 mo (9). Moreover, sdNVP has been demonstrated to be more effective than zidovudine chemoprophylaxis alone, and to be equivalent to NVP plus zidovudine in preventing MTCT (9-12). The efficacy of sdNVP in preventing MTCT is attributed to its potent antiviral activity, rapid absorption, placental transfer, and long half-life (13, 14). As a result, its use has been recommended by the World Health Organization as one of several options to reduce MTCT in resource-limited countries [Joint UNAIDS͞WHO press release (2000) (WHO, Geneva)].NVP is a potent nonnucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 (15-17). If administered as monotherapy, NVP selects for resistant variants within 1 wk, commonly those variants with the K103N and͞or Y181C mutations in reverse transcriptase, which confer cross-resistance to other approved drugs in the NNRTI class (18)(19)(20). The K103N mutation appears to have little effect on the replicative capacity o...

We determined the abilities of 10 technologies to detect and quantify a common drug-resistant mutant of human immunodeficiency virus type 1 (lysine to asparagine at codon 103 of the reverse transcriptase) using a blinded test panel containing mutant-wild-type mixtures ranging from 0.01% to 100% mutant. Two technologies, allele-specific reverse transcriptase PCR and a Ty1HRT yeast system, could quantify the mutant down to 0.1 to 0.4%. These technologies should help define the impact of low-frequency drug-resistant mutants on response to antiretroviral therapy.

Allele-specific RT-PCR quantified the emergence and decay of drug-resistant variants in patients over a broad range of frequencies (0.1-100%). The rate of decay of K103N variants after stopping NNRTI therapy was highly variable.

Antiretroviral therapy (ART) in human immunodeficiency virus type 1 (HIV-1)-infected patients does notclear the infection and can select for drug resistance over time. Not only is drug-resistant HIV-1 a concern for infected individuals on continual therapy, but it is an emerging problem in resource-limited settings where, in efforts to stem mother-to-child-transmission of HIV-1, transient nonnucleoside reverse transcriptase inhibitor (NNRTI) therapy given during labor can select for NNRTI resistance in both mother and child. Questions of HIV-1 persistence and drug resistance are highly amenable to exploration within animals models, where therapy manipulation is less constrained. We examined a pigtail macaque infection model responsive to anti-HIV-1 therapy to study the development of resistance. Pigtail macaques were infected with a pathogenic simian immunodeficiency virus encoding HIV-1 reverse transcriptase (RT-SHIV) to examine the impact of prior exposure to a NNRTI on subsequent ART comprised of a NNRTI and two nucleoside RT inhibitors. K103N resistance-conferring mutations in RT rapidly accumulated in 2/3 infected animals after NNRTI monotherapy and contributed to virologic failure during ART in 1/3 animals. By contrast, ART effectively suppressed RT-SHIV in 5/6 animals. These data indicate that suboptimal therapy facilitates HIV-1 drug resistance and suggest that this model can be used to investigate persisting viral reservoirs.

Background The contribution of low frequency drug-resistant HIV-1 variants to failure of antiretroviral therapy is not well-defined in treatment-experienced patients. Objective We sought to detect minor non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants at the onset of multidrug efavirenz-containing therapy in both NNRTI-naïve and NNRTI-experienced patients and to determine their association with virologic response. Methods Plasma samples at entry and virologic failure from patients enrolled in the AIDS Clinical Trials Group study 398 were analyzed by standard genotype, single-genome sequencing and allele-specific PCR (K103N and Y181C) to detect and quantify minor NNRTI-resistant variants. Results Minor populations of NNRTI-resistant variants that were missed by standard genotype were detected more often at study entry in NNRTI-experienced patients than NNRTI-naïve patients by both single-genome sequencing (8 of 12 vs. 3 of 15; P=0.022) and allele-specific PCR (>1% Y181C: 5 of 22 vs. 3 of 72, respectively, P = 0.016). K103N variants at frequencies >1% were associated with inferior HIV-1 RNA response to efavirenz-containing therapy between entry and week 24 (+0.5 vs −1.1 log10 copies/ml; P <0.001). Conclusions Minor NNRTI-resistant variants were more prevalent in NNRTI-experienced patients and were associated with reduced virologic response to efavirenz-containing multidrug regimens.

Antiviral resistance is a significant obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals. Because nonnucleoside reverse transcriptase inhibitors (NNRTIs) specifically target HIV-1 reverse transcriptase (RT) and do not effectively inhibit simian immunodeficiency virus (SIV) RT, the development of animal models to study the evolution of antiviral resistance has been problematic. To facilitate in vivo studies of NNRTI resistance, we examined whether a SIV that causes immunopathogenesis in pigtail macaques could be made sensitive to NNRTIs. Two simian-human immunodeficiency viruses (SHIVs) were derived from the genetic background of SIV mne : SIV-RT-YY contains RT substitutions intended to confer NNRTI susceptibility (V181Y and L188Y), and RT-SHIV mne contains the entire HIV-1 RT coding region. Both mutant viruses grew to high titers in vitro but had reduced fitness relative to wild-type SIV mne . Although the HIV-1 RT was properly processed into p66 and p51 subunits in RT-SHIV mne particles, the RT-SHIV mne virions had lower levels of RT per viral genomic RNA than HIV-1. Correspondingly, there was decreased RT activity in RT-SHIV mne and SIV-RT-YY particles. HIV-1 and RT-SHIV mne were similarly susceptible to the NNRTIs efavirenz, nevirapine, and UC781. However, SIV-RT-YY was less sensitive to NNRTIs than HIV-1 or RT-SHIV mne . Classical NNRTI resistance mutations were selected in RT-SHIV mne after in vitro drug treatment and were monitored in a sensitive allelespecific real-time RT-PCR assay. Collectively, these results indicate that RT-SHIV mne may be a useful model in macaques for the preclinical evaluation of NNRTIs and for studies of the development of drug resistance in vivo.

Background SARS-CoV-2 viral RNA (vRNA) is detected in the bloodstream of some patients with COVID-19 (“RNAemia”) but it is not clear whether this RNAemia reflects viremia (i.e., virus particles) and how RNAemia/viremia is related to host immune responses and outcomes. Methods SARS-CoV-2 vRNA was quantified by ultra-sensitive RT-PCR in plasma samples (0.5-1.0 ml) from observational cohorts of 51 COVID-19 patients including 9 outpatients, 19 hospitalized (non-ICU), and 23 ICU patients, and vRNA levels compared with cross-sectional indices of COVID-19 severity and prospective clinical outcomes. We used multiple imaging methods to visualize virions in pelleted plasma. Results SARS-CoV-2 vRNA was detected in plasma of 100%, 52.6% and 11.1% of ICU, non-ICU, and outpatients respectively. Virions were detected in plasma pellets by electron tomography and immunostaining. Plasma vRNA levels were significantly higher in ICU &gt; non-ICU &gt; outpatients (p&lt;0.0001); and for inpatient, plasma vRNA levels were strongly associated with higher WHO score at admission (p=0.01), maximum WHO score (p=0.002) and discharge disposition (p=0.004). A plasma vRNA level &gt;6,000 copies/ml was strongly associated with mortality (HR: 10.7). Levels of vRNA were significantly associated with several inflammatory biomarkers (p&lt;0.01) but not with plasma neutralizing antibody titers (p=0.8). Conclusions Visualization of virus particles in plasma indicates that SARS-CoV-2 RNAemia is due, at least in part, to viremia. The levels of SARS-CoV-2 RNAemia quantified by ultrasensitive RT-PCR correlate strongly with disease severity, patient outcome and specific inflammatory biomarkers but not neutralizing antibody titers.